The removal of introns from messenger RNA precursors by splicing is a mandatory step in the expression of most genes of higher eukaryotes. Previous work has defined a rare class of introns (U12-dependent introns) found in the genomes of most metazoan phyla. Members of this intron class are present in about 1 % of human genes including many with essential and/or disease-associated functions. Both the U12-dependent introns and the more abundant U2-dependent introns are spliced via a similar mechanism in large and complex ribonucleoprotein structures called spliceosomes. The two spliceosomes differ in their snRNA composition yet share significant similarities in the RNA-RNA interactions at the spliceosome core. These highly conserved sequences and interactions likely constitute the functional elements of the spliceosome. In addition, many but not all of the proteins involved in splicing appear to be shared between the two systems. A detailed understanding of the splicing signals and mechanism of this second splicing system is needed to understand the role of these introns in human gene expression and the biochemistry of splicing. This application proposes to examine the mechanism of splicing of this minor class of introns. First, RNA interference techniques will be used to screen for protein factors required for minor class splicing in Drosophila and mammalian cells. Second, experiments are proposed to investigate the specificity of spliceosome formation. RNA elements have been identified that control which spliceosomal RNAs interact with U12-dependent introns. The function of these elements and the factors they interact with will be investigated. Third, the critical functional features of minor class snRNAs will be identified including the exploration of relationships between these snRNAs and elements of self-splicing group II introns. Public health relevance: The regulated expression of genes is central to human growth, development, normal and pathological functioning and the response of the body to changes in the internal and external environment. An important point of gene regulation is the removal of introns from the primary transcripts of genes by RNA splicing. This proposal is designed to understand how the sites of splicing are correctly chosen and how the correct RNA fragments are linked together in the final mRNA products of human genes. [unreadable] [unreadable] [unreadable]